Evident in its name, Helicobacter pylori is a helical bacterium that colonizes the human stomach causing clinical outcomes ranging from mild gastritis to peptic ulcer and gastric cancer. Descriptions of H. pylori virulence include a widely accepted hypothesis that the bacterium's spiral shape enhances its ability to swim through the viscous epithelial mucus layer and successfully colonize the stomach. This theory, however, has never been tested and the molecular determinants of H. pylori's shape remain unknown. Using a visual screen of a transposon mutant library, we identified clones displaying two classes of altered cell shape: straight rod and curved vibrioid. Genetic analyses revealed three chromosomal loci, one with at least three genes all affecting cell shape. Biochemical analyses indicate that some of these proteins modify the cell wall peptidoglycan (PG) structure. All mutants tested show slight, but reproducible defects in halo formation in soft agar suggesting compromised swimming or chemotaxis. One vibrioid and two rod-shaped mutants were tested for their ability to colonize the mouse stomach and all showed an altered phenotype compared to wild type or complemented strains. Interestingly the rod-shaped mutants colonized better than their spiral counter parts while the vibrioid mutant showed lower colonization. Thus the identified spiral shape-determining genes influence stomach colonization, but how and why remains to be elucidated. We have two goals with the current grant proposal. The goal of Aim 1 is to understand how the gene products identified in our loss of spirality screen alter cell shape. What is the role of the cell wall? Do the gene products identified within or between shape classes interact? Do they interact with known PG biosynthetic and degrading enzymes or with bacterial cytoskeletal elements? The goal of Aim 2 is to understand how these proteins function during stomach colonization. Are the colonization phenotypes due to altered swimming as previously hypothesized, or is chemotaxis altered? Are the identified cell wall modifications important for the bacteria's ability to survive stresses encountered in the stomach such as fluctuations in osmolarity, acidity and exposure to antimicrobial peptides? Do the cell wall alterations influence PG signaling and/or detection by the host immune system? We believe this work will yield significant advancements in both cell biology and pathogenesis. An outstanding question in microbial cell biology is how and why spiral shape is maintained. The discovery that some of these proteins modify PG is also significant from both cell biology and pathogenesis perspectives. H. pylori's PG activates a major pro-inflammatory pathway via the intracellular pathogen recognition molecule Nod1. Recognition of specific PG components by epithelial cells has been shown to be important for both symbiotic and pathogenic interactions of bacteria and their host. Altogether the experimental aims will probe how and why H. pylori maintains its spiral shape and its role in pathogenesis. PUBLIC HEALTH RELEVANCE: Evident in its name, Helicobacter pylori is a helical bacterium that colonizes the human stomach causing clinical outcomes that range from mild gastritis to peptic ulcer and gastric cancer. We discovered genes that promote spiral shape by this bacterium can alter its cell wall. Study of how these cell wall modifications promote spiral shape and bacterial survival in its unique niche will help us better understand how this bacteria causes disease and discover new ways to inhibit infection.